Biology subjects

Chai, T.

Publications and source records attributed to Chai, T..

6 recordsLinked to original sources

bioRxiv2026

Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

A preeminent goal of virology is to discover cellular genes that mediate virus entry. Genome-wide loss-of-function screens can illuminate single genes necessary for virus entry, but are stymied by genetic redundancy. Here we report a genome-wide CRISPR activation screening strategy to discover single genes that are sufficient for viral entry into normally-uninfectable cells. Sequential rounds of viral infection vastly enhanced screening sensitivity. This sequential screening strategy was generalizable to two unrelated viruses--Ebola and rabies viruses--and could broadly accelerate the discovery of viral entry factors.

microbiology↗

bioRxiv2024

TMX1, a disulfide oxidoreductase, is necessary for T cell function through regulation of CD3zeta

T cell-targeted therapies are commonly used to manage T cell hyperactivity in autoimmune disorders, graft-versus-host diseases (GVHD), and transplant rejections. However, many patients experience significant side effects or inadequate responses to current treatments, highlighting the urgent need for alternative strategies. In this study, we searched for regulators of T cells through proximity labeling with APEX2 to detect proteins interacting with CD8, a coreceptor of the T-cell receptor (TCR). This screen revealed TMX1, an ER resident transmembrane disulfide oxidoreductase, is essential for T cell cytotoxicity and NFAT, NF{kappa}B, and AP1 signaling but not cell proliferation. TMX1 deletion decreases surface TCR expression and destabilizes CD3{zeta}, a subunit of TCR complex; however, overexpression of CD3{zeta} rescues the phenotype, suggesting that TMX1 is not required for CD3{zeta} function. Mechanistically, TMX1 was found to directly engage the CxxC motif of CD3{delta}, which has been reported to be essential for proper TCR assembly and function. We hypothesize that the loss of TMX1 interaction with CD3{delta} leads to impaired TCR assembly and subsequent CD3{zeta} destabilization. These findings identify TMX1 as a novel regulator of T-cell receptor assembly and a potential target for immunosuppressive therapy.

immunology↗

bioRxiv2024

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies and translating this success to other cancers remains an ongoing clinical objective. Next-generation T cell products in development aim to genetically modulate many facets of cell behavior, for which gene-nominating platforms provide a useful framework for prioritization. Among competing screening approaches, CRISPR activation (CRISPRa) technology permits gain-of-function (GoF) gene surveys at genome-wide scale, but routine implementation in primary T cells has been stymied by high cell requirements ([~]107 - 108) and abbreviated activity. Here, we describe a novel cell manufacturing schema using an all-in-one transposon-based gene delivery system coupled with CAR-restricted cell expansion to generate yields (109) of primary T cells bearing CAR and CRISPRa transgenes that are well above the threshold needed for genome-scale screening. CRISPRa activity is sustained via the inclusion of divergent, duplicate Elongation Factor 1 core/human T-cell leukemia virus (EF1-HTLV) hybrid promoters; while guide RNA representation is preserved through late lentiviral transduction, thus preventing bottlenecking and premature candidate pruning. CRISPRa-CAR T cells manufactured via this pipeline retain potent on-target gene-overexpression (>85% target+) across varied cell subsets (e.g. Tim-3+Lag3+ or serial-challenge) and timescales (>14 days). When deployed to survival-based genome-wide selection landscapes, CRISPRa-CAR pools nominate known and novel endogenous genes capable of enhancing CD8+ CAR T survival in cytokine-rich (e.g. MYC, FUT6, IRF4, GSE1) and cytokine-depleted (e.g. CSF2RB, STAT6, IRF4, GSE1) settings of tumor challenge. This system will have broad utility for therapy-enhancing gene discovery.

immunology↗

bioRxiv2020

Spatial coupling between DNA replication and mismatch repair in Caulobacter crescentus

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL displays an apparently more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the {beta}-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.

microbiology↗

bioRxiv2020

CD47 prevents the elimination of diseased fibroblasts in scleroderma

Scleroderma is a devastating fibrotic autoimmune disease. Current treatments are partly effective in preventing disease progression, but do not remove fibrotic tissue. Here, we evaluated whether scleroderma fibroblasts take advantage of the "dont-eat-me-signal" CD47 and whether blocking CD47 enables the bodys immune system to get rid of diseased fibroblasts. To test this approach, we used a Jun-inducible scleroderma model. We first demonstrated in patient samples that scleroderma upregulated JUN and increased promotor accessibilities of both JUN and the CD47. Next, we established our scleroderma model demonstrating that Jun mediated skin fibrosis through the hedgehog-dependent expansion of CD26+Sca1-fibroblasts in mice. In a niche-independent adaptive transfer model, JUN steered graft survival and conferred increased self-renewal to fibroblasts. In vivo, JUN enhanced the expression of CD47, and inhibiting CD47 eliminated an ectopic fibroblast graft and increased in vitro phagocytosis. In the syngeneic mouse, depleting macrophages ameliorated skin fibrosis. Therapeutically, combined CD47 and IL6 blockade reversed skin fibrosis in mice and led to the rapid elimination of ectopically transplanted scleroderma cells. Altogether, our study is the first to demonstrate the efficiency of combining different immunotherapies in treating scleroderma and provide a rationale for combining CD47 and IL6 inhibition in clinical trials.

immunology↗

bioRxiv2019

IL-1RA controls the development of esophageal cancer by inhibition of VEGF via PI3K/NF-κB signaling pathway

IL-1RA has been reported to function as tumor suppressor in a variety of tumors, but its function and mechanism in esophageal cancer remains largely unknown. Our previous studies have shown that IL-1RA is downregulated in primary esophageal cancer(EC), and this downregulation of IL-1RA is closely related to TNM staging and survival prognosis. In this study, we observed that over-expression of IL-1RA inhibits proliferation, migration, and tumor growth in esophageal cancer cells(ESCC), inhibits the expression and secretion of VEGF, and inhibits tumor angiogenesis. Further studies have shown the overexpression of IL-1RA inhibits the transmission of PI3K/NF-kappaB. Taken together, this study shows that IL-1RA can regulating the development of esophageal cancer by inhibiting VEGF expression and secretion via inhibiting PI3K/NF-kappaB signaling, and it may serve as a potential prognostic marker and therapeutic target for EC.

cancer biology↗